On account of the clearance by kidney, liver and other unclear factors, most of the therapeutic bioactive peptides/proteins are often rapidly cleared in body, and their half-life generally ranges from only a few minutes to several hours. During therapeutic treatment, larger dosage and frequent injections are required to maintain effective drug concentration, which does not only mean a lot of pain to patients, but also decreases curative effect and increases the toxicity due to the fluctuation of plasma concentration.
Today, there are several ways have been reported to extend the half-life of bioactive peptides/proteins in vivo. Such as modifying bioactive peptides/proteins with water-soluble polymers (e.g., polyethylene glycol, dextran, etc.), which has been successfully applied, like PEG-ADA, PEG-IFNα and so on. The modification could prolong the half-life in vivo, increase stability and solubility, reduce immunogenicity and so on. But still there've been lots of problems with these modifications. First of all, the bioactivity of the proteins/peptides modified with chemical are generally decreased significantly or even inactivated (Veronese F M, Biomaterials, 22:405-417, 2001). Second, the way the polymers connecting to proteins/peptides is generally by forming covalent bond with amidogen, hydroxyl radical, imidazolyl radical and/or other chemical groups located on the surface of the proteins/peptides. However, most of the proteins/peptides have large molecule weight and complex structure. There would be multiple potential groups that may reactive with activated PEG. So the stability, bioactivity and/or other characters of the product varied if different sites are linked to PEG. What is more, most of the chemical synthetic polymers, such as PEG, can not be degraded by organisms. For example, it has been found that long-term and high-dose injection of PEG-interferon (PEG-IFNα2a) would accumulate in the kidney (Conover C D et al., Artificial Organs., 21:369-3 78,1997; Bendele A et al., Toxicol Sci., 42:152-157, 1998). From drug design perspective, drugs without accumulation would clearly be more secure. On the other hand, PEG-modified proteins have been found to produce a PEG antibodies (defined as multivalent hapten) which consequently affect the half-life of the drugs (Caliceti P & Veronese F M, Adv Drug Deliv Rev., 55:1261-1277, 2003).
Because of these technical problems, although it has been available to improve the pharmacokinetic profile of proteins/peptides in vivo by chemical modification for a long time, there are few chemical modified proteins/peptides been used in clinical practice.
It's also available to improve the stability of proteins/peptides in vitro and to prolong its half-life in vivo by fusion with some specific carrier protein. As described in the U.S. Pat. Nos. 5,876,969 and 5,766,88 and 7,176,278, the half-life of bioactive peptides/protein was improved after fusion with albumin, Fc fragment of antibody, transferrin (fragment/mutant of transferrin). The mechanism why these fusion proteins can extend the half-life is attributing to carrier protein's long in vivo half-life. To be a perfect carrier protein should have the following characteristics: 1. longer half-life in vivo; 2. Non-immunogenicity; 3. without biological function that unrelated to extending the half-life; 4. Not affect the bioactivity of the fused therapeutic proteins. However, there are no any solutions in public that may meet all of the above requirements until now. The first problem is the increase of immunogenicity, such as Fc fragment, of which the structure is not conservative. It is easy to cause the immune response because of the diverse sequence and structure. In addition, these carrier proteins usually have some biological effects, for example, Fc fragment can bind to complement (Fc receptor) to cause allergy, phagocytosis regulation, cell damage effect, etc. HSA usually partakes in the transport and metabolism of many substances. For a carrier protein, the existence of these biological characteristics is negative. Moreover, these carrier proteins themselves have complex spatial structure, which would decrease the activity of the fused bioactive protein due to the steric effect (Baggio L L et al., Diabetes., 53: 2492-2500, 2004; Huang Y S et al., Eur J Pharm Biopharm.,67:301-308, 2007).
In summary, the existing technology to extend the in vivo half-life of therapeutic proteins have the following drawbacks: 1. heterogeneous products, complex technological requirements; 2. what used to modify the protein cannot be degraded by organism, and would accumulate in vivo; 3. to increase the immunogenicity; 4. resulting in a significant reduction or even complete loss of the bioactivity of fused proteins; 5. may bring in unneeded side biological effect. Neither chemical modifications, nor fusion with carrier protein can completely avoid the above disadvantages.
In order to avoid the disadvantages of these natural carrier protein like albumin or Fc fragment, the artificial amino acid sequences has been tried as a carrier protein. David W. Leung etc. artificially synthesized poly-glutamate as a fusion carrier to prolong the half-life of protein drugs (US 20080176288). Synthetic poly-glycine has also been tried as a fusion vector (Schlapschy M et al., Protein Eng Des Sel., 20:273-284, 2007). There are other fusion vectors artificially synthesized with hydrophilic amino acids(like Gly, Asp, Glu, Ser, etc al) in alternative to extend the half-life of protein drugs as well. However, it's complicated to predict actual effects of the completely artificially synthetic fusion carriers. There would be many problems. For example, 1. Due to the complex relation between structure and the function of protein, it is difficult to predict the actual high order structure (such as secondary and tertiary structure) of the one synthesized exactly to be the one designed, and so it is difficult to predict the potential biological activity and immunogenicity; 2. Artificially designed repetitive sequences, especially those highly repetitive ones, are often different from the natural developed ones and are hard to be expressed because the actual expression levels are often too low to apply in practice. The inventor have tried to construct a recombinant poly-Glu as a fusion carrier to extend the half-life of protein drugs according to David W. Leung, etc. (US20080176288), but it was impracticable actually.
Therefore, it's urgently needed to develop a simple and effective technological solution that can improve the residence time of protein/peptides both in vitro and in vivo and with little or none side effects.